6.3 Flashcards
What is DNA sequencing?
A technique that allows genes to isolated and read.
Explain the steps of pyrosequencing.
1) DNA is mechanically cut in fragments of 300-800 base pairs using a nebuliser
2) DNA is then degraded into single strands of DNA (ssDNA) which will be used as a template and these are immobilised.
3) Primer sequence is added to DNA as well as enzymes:
- Apyrase
- Luciferase
- ATP Sulfurylase
- DNA polymerase
and the substrates:
- Adenosine-5 phosphosulfate (APS)
- Luciferin
And activates nucleotide’s, ATP, TTP, CTP and GTP.
4) The activated nucleotide’s have two extra phosphates which are released when they bind to the single strand of DNA.
The two phosphates released are called pyrophosphate.
The presence of APS causes the ATP sulfurylase to convert pyrophosphate into ATP.
Presence of ATP causes luciferase to convert luciferin into oxyluciferin generating light which is detected by a camera
5) The intensity of light is proportional to the number of ATP present therefore light intensity can be used to identify number of activated nucleotide’s that bind to DNA strand.
What is bioinformatics?
Analysing and interpreting biological data on genes.
What is the purpose of the human genome project?
To sequence the human genome
What have we learned from comparing genome of species?
- Some genes become co-opted through evolution
- Regulatory regions of genes change through evolution changing number of proteins produced
How has genome comparisons been used to study evolutionary relationships?
- Bones and teeth from fossil can be used to study animals evolutionary history
How has genome comparison helped in understanding variation between individuals?
- Methylation affects expression of genes
Mapping methylation of genome helps identify
possible causes of disease and cancer.
Study called epigenetics - Mutations which may cause changes to protein structures and expression
What is synthetic biology?
Designing and building of useful biological devices which are able to:
- provide food,
- enhance environment,
- process and store biological data
- maintain human health
How has gene sequencing allowed for the
sequences of amino acids in polypeptides to
be predicted?
If human genome is already mapped and the gene that codes for protein is known then the amino acid sequence can be identified.
How is DNA profiling carried out?
1) DNA sample is taken via a swab
2) DNA is digested using restriction enzymes which cut DNA at specific recognition points into varying lengths of DNA
3) Fragments then undergo electrophoresis and are stained.
4) Band pattern will be seen
5) Band pattern compared to other individuals DNA which undergoes exact same process.
What are short tandem repeats?
Highly variable short lengths of repeating DNA sequence.
STR is polymorphic but the number of alleles shared in a gene pool is small.
What are the application of DNA profiling?
- Identify remains
- Identify victim body parts
- Identify blood relations
- Identify Nazi war criminals
- Settle maternity and paternity disputes
Explain the process of PCR (Polymerase Chain Reaction)
1) DNA is mixed with magnesium ions, Taq Polymerase, nucleotide’s and a primer
2) DNA mixture heated to 94-96 in order to break hydrogen bonds between nitrogenous bases creating two single strands of DNA
3) Mixture cooled to 68 to allow primer to attach/bind/anneal to single strands of DNA. Causing part of DNA single strand to become double stranded.
4) Heated to 72 as this is the optimum temperature for Taq Polymerase to function and keeps strands separate
5) Taq polymerase attaches to primer and catalyses addition of nucleotide’s from the 5’ to 3’ direction
6) Once it has reached the other side the process will repeat
What are the applications of PCR?
- Tissue Typing of donor and recipient before transplant to check risk of rejection
- Oncogenes cause cancer, If these are identified they can be used to tailor medication
- Mutations in DNA. Recessive allele of parents, fetal cells from parent blood stream for pre-natal embryonic screening, 1 cell from 8 cell embryo to check for mutations before implantation (IVF)
- Research of fossils, DNA from their bones and teeth
- Forensic evidence left behind at crime scene to identify perpetrators
- Identify viral infection as virus DNA can be detected amongst host cell DNA (diagnosis of HIV)
- Spread of disease can be controlled by checking who have contracted disease and also any mutations in the disease.
How is electrophoresis carried out?
1) DNA sample is cut into fragments using restriction enzymes which cut DNA at specific recognition sites at 35-40 degrees for an hour.
2) Agarose gel is made and poured into central part of tank whilst combs in place at one end. After gel set, buffer solution poured covering agarose gel and ends of tank. After this combs are removed leaving wells at one end.
3) A loading dye is added to tubes containing digested DNA
4) DNA mixture added to wells using pipette. hold pipette just above well in buffer solution. Density of loading dye causes DNA to fall to bottom of well
5) Electrodes are added and connected to 18v battery for 6-8 hours. Higher voltage can be used but must be limited to 5mA as risk of electric shock.
6) DNA fragments move at different speeds, smaller fragments move faster travelling further away
7) Buffer solution poured out, dye added which adheres to DNA staining it.
